Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity
Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity
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DOI：
10.1089/crispr.2022.0065
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发表时间：
2022-11-15
期刊：
影响因子：
3.7
通讯作者：
Royzen, Maksim
中科院分区：
文献类型：
--
作者：
Hoy, Alyssa;Zheng, Ya Ying;Royzen, Maksim
CRISPR-Cas9系统是一种重要的基因组编辑工具，在治疗人类遗传病方面具有巨大的潜力。CRISPR技术的临床成功依赖于将修饰整合到单引导RNA(SgRNA)中。然而，化学合成长度超过100个核苷酸的修饰sgRNAs是困难和低产率的。我们开发了一种结合策略，利用生物正交化学有效地组装包含核苷酸碱基修饰的功能性sgRNAs。所描述的方法需要化学合成两个较短的RNA寡核苷酸：含有四嗪(Tz)基团的31-聚体和带有反式环辛烯(TCO)部分的70-聚体。将这两个寡核苷酸连接形成功能性sgRNAs。采用双组分连接方法合成了含有N-1-甲基腺苷(m(1)A)、N-6-甲基腺苷(m(6)A)、2-硫代尿苷(S(2)U)和4-硫代尿苷(S(4)U)等碱基修饰的sgRNA库。在体外和表达Cas9的HEK293T细胞中，研究了这些RNA修饰对CRISPR总活性的影响。
The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N-1-methyladenosine (m(1)A), N-6-methyladenosine (m(6)A), 2-thiouridine (s(2)U), and 4-thiouridine (s(4)U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.